Figure 2.

miR-483-5p does not directly bind to TAU mRNA or impact TAU levels. (A) Scheme represents the predicted binding site for miR-483-5p in the 3′-UTR of the TAU mRNA. (B) Prediction indicates human-specific miR-483-5p binding site in 3′-untranslated region (3′-UTR) of the TAU mRNA among different species. (C) Quantitative reporter assay for luciferase activity in HEK293 cells. Reporter constructs carrying a single binding site (CTR, WT, MUT, PM) for miR-483-5p were tested. The miRNA activity on four pmirGLO constructs was measured simultaneously: an empty pmirGLO vector (CTR), a wild-type potential binding site for miR-483-5p (WT), a mutated binding site (MUT), and a site with full complementarity (PM). Interaction scheme of WT and MUT binding site of 3′-UTR of the TAU mRNA with miR-483-5p are shown below. PM (not shown here) is designed to perfectly match the miR-483-5p. Firefly luciferase activity was normalized against renilla luciferase activity. Data are shown as mean ± standard error from four independent experiments. One way ANOVA with post-hoc Tukey HSD was used for statistical analysis. (D) RT-qPCR analysis of TAU mRNA levels upon miR-483-5p mimic transfection in SK-N-MC cells. GAPDH was used as a reference. Data are shown as mean ± standard error from three biological replicates; Unpaired Student’s t-test was used for statistical analysis. In all cases, annotations were made on plots only where significant differences (p < 0.05) were found.